Abstract/Project Summary Non-alcoholic fatty liver disease (NAFLD) is now the most common chronic liver disease worldwide. However, its pathophysiology is unresolved and treatment options are lacking. Poor maternal diet and obesity promote NAFLD in offspring, but the mechanisms by which this occurs are not clear. We propose to identify the impact of maternal western style diet (WSD) on offspring immune cell and liver phenotypes in a non-human primate (NHP) model with features similar to human pediatric NAFLD. While the pathophysiology of NAFLD is multifactorial, pro-inflammatory macrophage (M?) activation and recruitment of bone marrow monocytes to the liver are critical for its progression. The innate immune system is derived from hematopoietic stem cells (HSCs), which reside primarily in the bone marrow. Maternal diet has been shown to skew HSC function, which may manifest as alterations in M? development and activity, potentially promoting NAFLD progression. We find that maternal WSD in our NHP model increases fetal steatosis (liver fat), decreases proliferation of bone marrow cells and causes a proportional increase in myeloid (innate immune) cell production. Strikingly, these phenotypes are also present in juvenile NHP exposed to maternal WSD then weaned onto a healthy diet for the remainder of life. In addition, we observe dysregulated M? cytokine response in NHP fetuses exposed to maternal WSD. Finally, hypoxia-inducible factor (HIF)-1a has emerged as a critical driver of the M? pro-inflammatory phenotype. Our preliminary data in mice show that mice fed a WSD have increased hepatic M? HIF1A RNA expression and HIF-1a protein stabilization has been shown to promote M? inflammation and fibrosis in mice. This suggests that inhibiting HIF1a in bone marrow may prevent inflammatory M? activity in NAFLD. Therefore, current data supports the novel hypothesis that maternal WSD differentially programs HSCs in utero to promote persistent myeloid cell skewing and downstream M? dysfunction that ultimately drives liver damage and fibrosis later in life, and that Hif1a is necessary for this M? dysfunction. To investigate this hypothesis we propose to: 1) We will utilize qPCR and microscopic techniques in livers from fetal and juvenile NHPs to determine the impact of maternal WSD on fibrosis, tissue inflammation and lipid content. We will also investigate the transcriptional phenotypes and proportions of recruited vs. resident M? in livers, to determine the relative contributions of these cells in promoting NAFLD. 2) Identify how maternal WSD alters transcriptional pathways in HSCs responsible for shifts in HSC development, proliferation, and M? phenotypes in NHP offspring, including M? cytokine response. 3) We will test whether deletion of Hif1a in hematopoietic cells in mice is necessary for maternal WSD induced alterations in pro-inflammatory hepatic M? response and fibrosis. These studies will give descriptive and mechanistic insight into how maternal WSD impacts offspring NAFLD, which may lead to new therapeutic approaches to hinder NAFLD progression or attenuate the impact of this prevalent disease.